Study on morphological changes and function of long-term cultured primary rat hepatocytes in a sandwich configuration

Abstract

Objective To study the morphological changes and repolarization and function of the long-term cultured primary rat hepatocytes in a sandwich configuration.Methods Hepatocytes were isolated by a two-step collagenase perfusion based on a procedure described by Seglen and modified by us.Primary rat hepatocytes were cultured in a sandwich configuration.The morphological changes were observed under an inverted microscope.The redistribution of specific membrane protein was de-tected by fluorescent immunocytochemistry.Hepatocyte albumin mRNA were examined by RT-PCR.The hepatic function was also examined.Results Routinely,200-300 million cells were obtained per liver,with viabilitv and purity ranging from 90% to 96% and 96% to 99%.After being cultured for 6-8 hours,the hepatoeytes formed a hepatic cordes automatically.On the third day,the expression of albumin mRNA recovered dramatically.On the fourth day,the DPPIV was mainly concentrated at the canalicular front.Five days later,a canalieular network were completed.Hepatocyte polygonal mor- phology lasted for more than 49 days.Conclusion Sandwich configuration more closely mimics the normal histological organization of the liver and hepatocytes cultured in this configuration may maintain its morphology and function for a long period. Key words: Cells,cultured; Sandwich; Hepatocyte; Primary culture; Repolarization ; mRNA